1. Field of the Invention
This invention relates to the fields of immunology and leukemia therapy. More specifically, this invention relates to the use of synthetic analogue peptides to induce heteroclitic human T cell responses against native peptides of the synthetic analogues.
2. Description of the Related Art
Chronic myelogenous leukemia (CML) is a pluripotent stem cell disorder characterized by the presence of the Philadelphia chromosome (Ph). The Philadelphia chromosome represents a translocation in which the c-abl oncogene has moved from chromosome 9 into the breakpoint cluster region (bcr) within the bcr gene on chromosome 22, resulting in a chimeric bcr-abl gene. The fused genes encode an 8.5 kb chimeric mRNA which is usually translated into a 210-kDa or 190-kDa protein. This bcr-abl protein is a tyrosine-kinase which is uniquely present in the leukemia cells of chronic myelogenous leukemia patients and is necessary and sufficient for transformation.
In chronic myelogenous leukemia, the breakpoint in the bcr gene occurs either between bcr exon 2 (b2) and 3 (b3) or between bcr exon 3(b3) and 4(b4). Although aberrant bcr-abl fusion genes and bcr-abl mRNA splicing can occur, the majority of patients with chronic myelogenous leukemia therefore express p210-b3a2 or p210-b2a2; often both p210 and p 190 proteins are expressed together with low levels of p 190-e 1 a2 bcr-abl proteins. In Ph1 positive acute lymphocytic leukemia (ALL), the predominant breakpoint is at the e1a2 site.
The chimeric fusion proteins are potential antigens. First, the proteins are uniquely expressed in chronic myelogenous leukemia cells in which the junctional regions contain a sequence of amino acids that is not expressed on any normal protein. Secondly, as a result of the codon split on the fused message, a new amino acid, lysine in b3a2, and a conserved amino acid, glutamic acid in b2a2, is present at the exact fusion point in each of the proteins. Therefore, the unique amino acid sequences encompassing the b3a2 and b2a2 breakpoint region can be considered truly tumor specific antigens. Despite the intracellular location of these proteins, short peptides produced by cellular processing of the products of the fusion proteins can be presented on the cell surface within the cleft of HLA molecules and in this form they can be recognized by T cells.
Recent clinical trials demonstrated that a tumor specific, bcr-abl derived multivalent vaccine may be safely administered to patients with chronic phase chronic myelogenous leukemia. The vaccine reliably elicits a bcr-abl peptide-specific CD4 immune response as measured by DTH in vivo, by CD4+ T cell proliferation ex vivo and by gamma interferon secretion in an ELISPOT assay. However, no CD8 responses in HLA A0201 patients and only weak responses in HLA A0301 patients were detected using a sensitive gamma interferon ELISPOT assay.
Wilms tumor protein 1 (WT1) is a zinc finger transcription factor expressed during normal ontogenesis such as in fetal kidney, testis and ovary. In adults, WT1 expression is limited to low levels on hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes and some cells in testis and ovary. Recent demonstration that WT1 is overexpressed in several types of leukemia suggested that WT1 would be an attractive target for immunotherapy. Three peptide nonamers from WT1 have been identified to generate a WT1 specific cytotoxic response in the context of HLA 0201 and HLA 2402. However, as WT1 protein is a self-antigen, breaking tolerance is a potential concern.
For stimulation of responses the strength of CD8 responses depends upon the binding affinity of the target peptide to class I MHC molecules, the peptide-HLA complex stability, and the avidity of the T cell receptor binding for the peptide complex. Killing of native CML cells also requires adequate processing and presentation of the natural antigen. Therefore the lack of reproducible CD8 responses in these clinical trials could be the result of the biochemistry of these class I peptide-HLA interactions, which results in their weak immunogenicity to cytotoxic CD8 cells. None of the native CML peptides reported to bind to human MHC bound the HLA pocket with high affinity. This may explain, in part, the lack of a detectable immune response to bcr-abl peptides as proteins seen in patients with chronic myelogenous leukemia despite the appearance of this antigen in the CML cells.
In some antigenic systems peptide analogues are used to circumvent a poor immunogenic response. A high correlation has been found between overall analogue peptide affinities for MHC class I molecules and in vivo peptide immunogenicity in HLA-A2Kb transgenic mice. A better correlation with a peptide's ability to form stable HLA-A0201 complexes and immunogenicity has been reported. Improved immunogenicity in HLA-A0201/Kb transgenic mice also has been reported for analogues of a self-peptide, gp100154-162, displaying both higher affinity and more prolonged complex stability than the natural peptide.
To design peptide analogues several successful algorithms have been utilized in which large protein sequences are scanned for the presence of suitable binding motifs, leading to the identification of predicted antigens that have subsequently been experimentally validated. Analogs of antigenic peptides have been formulated by direct modifications of MHC anchor positions, which are referred to as “MHC anchor-modified ligands”, or modifications of TCR contact sites, which generally are termed “altered peptide ligands”. The identification of peptide epitope analogues that strengthen the stability of the MHC-peptide complex in vivo and in vitro is thought to enhance the potency of intrinsically weak immunogenic peptides for the activation and amplification of relevant T-cell subsets. This concept was originally described in a murine CD4+ T cell model using HIV peptides (1), and now has been extended to a variety of viral and tumor immunological systems.
Artificial variants of MHC class I-binding self-peptides have been designed (2). Since these variant peptides were foreign to the host immune system, a strong CTL response was induced. Unlike weak T cell responses to self-peptide-MHC complexes, CTL responses to variant peptides can be sustained for a longer period without causing annihilation of the clones due to insufficient signals for cell division or survival. Since a substantial fraction of such CTLs cross-react with non-mutated self-peptides expressed in tumor cells in much smaller amounts, immunization with variant peptides may be a more efficient method to induce CTLs against tumors. The scoring system for MHC class I-binding peptides should provide a convenient method for design of cross-reactive self-mimicking peptides for immunization.
The improved immunogenicity in vivo and relevance of MHC anchor-modified ligands was first shown formally in human neoplastic disease in a controlled study of patients with malignant melanoma using a melanoma-associated A0201 restricted peptide derived from gp100. It has been shown recently with HLA-tetramer based detection methods that the parental Melan-A anfigenic peptides are weak agonists which activate antigen-specific T cells suboptimally (3). In contrast, melan A peptide analogues were identified that behaved as full agonists and induced full T cell activation leading to strong tumor antigen-specific CTL responses (4).
Simple motifs and the statistical binding matrices can be used to perform a crude search for MHC-binding peptides. Unfortunately, the presence of a simple sequence motif does not correlate well with binding. Therefore these simple motifs are not always necessary or sufficient for binding. Only 30% of the peptides that carry such simple motifs bind well when examined in a biochemical binding assay. Predictions of binding can be improved considerably when extended motifs are used, rather than the simple motifs. About 70% of the peptides carrying an extended motif bind well.
Assuming that each amino acid in each position contributes a certain binding energy independent of the neighboring residues and that the binding of a given peptide is the result of combining the contributions from the different residues, multiplying the relevant matrix values should give an indication of the binding of the corresponding peptide. Such statistical matrix-driven predictions have been somewhat more successful, thereby suggesting that MHC binding is to some extent the result of a combinatorial specificity. The identification of analogues peptides based on these methods has been applied recently to the identification of CTL epitopes deduced from proteinase 3, melanoma antigen 3, mucin 1 and telomerase.
The weak immunogenicity of native bcr-abl fusion peptides, as demonstrated by poor lysis of the cells, or the problem of tolerance using native peptides from a self-antigen, such as WT1, has prevented use of these native peptides as an effective vaccine against CML. A need exists in the art to develop therapeutic strategies using vaccination against a truly tumor specific antigen that is also the oncogenic protein required for neoplasia. There is a need for improved synthetic peptide analogues designed to elicit a greater immunogenic response.
The prior art is deficient in the lack of synthetic analogue peptides that could generate an immune response that not only recognizes the immunizing epitopes, but that also cross reacts with the original native peptides. Specifically, the prior art is deficient in synthetic peptide analogs with both improved HLA binding and improved ability to elicit a greater immunogenic response against cancer cells. The present invention fulfills this longstanding need and desire in the art.